CNS4 causes subtype‐specific changes in agonist efficacy and reversal potential of permeant cations in NMDA receptors

Abstract The NMDA subtype of glutamate receptor serves as an attractive drug target for the treatment of disorders evolving from hyper‐ or hypoglutamatergic conditions. Compounds that optimize the function of NMDA receptors are of great clinical significance. Here, we present the pharmacological characterization of a biased allosteric modulator, CNS4. Results indicate that CNS4 sensitizes ambient levels of agonists and reduces higher‐concentration glycine & glutamate efficacy in 1/2AB receptors, but minimally alters these parameters in diheteromeric 1/2A or 1/2B receptors. Glycine efficacy is increased in both 1/2C and 1/2D, while glutamate efficacy is decreased in 1/2C and unaltered in 1/2D. CNS4 does not affect the activity of competitive antagonist binding at glycine (DCKA) and glutamate (DL‐AP5) sites; however, it decreases memantine potency in 1/2A receptors but not in 1/2D receptors. Current–voltage (I‐V) relationship studies indicate that CNS4 potentiates 1/2A inward currents, a phenomenon that was reversed in the absence of permeable Na+ ions. In 1/2D receptors, CNS4 blocks inward currents based on extracellular Ca2+ concentration. Further, CNS4 positively modulates glutamate potency on E781A_1/2A mutant receptors, indicating its role at the distal end of the 1/2A agonist binding domain interface. Together, these findings reveal that CNS4 sensitizes ambient agonists and allosterically modulates agonist efficacy by altering Na+ permeability based on the GluN2 subunit composition. Overall, the pharmacology of CNS4 aligns with the need for drug candidates to treat hypoglutamatergic neuropsychiatric conditions such as loss function GRIN disorders and anti‐NMDA receptor encephalitis.

NMDA receptor dysfunction is a major cause for the pathogenesis of psychiatric disorders, in addition to the well-studied dopamine hypothesis. 23,24 There are genetic, biochemical and pharmacologic lines of evidence supporting the contribution of NMDA receptor hypofunction to the pathophysiology of schizophrenia 25 and the comorbidity of substance abuse. 26 Further, it was recently estimated that one in 5026 people in the United States could have a rare developmental neuropsychiatric condition recently recognized as GRIN (Glutamate Receptor Ionotropic NMDA) disorder. 27 Majority of the GRIN patients are less than 12-year-old. The clinical presentation of GRIN disorders include ADHD, developmental delay, intellectual disability, epilepsy, autism spectrum disorders and movement disorders. 28 Currently, there is no cure for this condition. GRIN disorder-causing amino acid mutations in the polypeptide chains of NMDA receptor subunits are reported in the Center for Functional Evaluation of Rare Variants (CFERV) database. According to details provided at CFERV, most patients have loss-of-function mutations leading to hypoglutamatergic neurotransmission. Therefore, drugs that selectively modulate hypoactive NMDA receptors could be used to treat GRIN disorders with reduced glutamate potency. In this study, we performed pharmacological characterization of a small molecule (CNS4) that modulates NMDA receptor function based on GluN2 subunit composition and agonist concentration.

| Dose-response curves
Electrophysiological responses were measured using a standard two- agonist doses were applied on the oocytes using an 8-channel perfusion system (Automate Scientific), and the responses were digitized for quantification (Digidata 1550A and pClamp-10, Molecular Devices). All data points were obtained from the steady state currents unless stated otherwise. Dose-response relationships were fit to an appropriate curve-fitting equation using GraphPad Prism-7.
Non-linear regression was used to calculate EC 50 , IC 50 , and fold change or percentage maximal response. Curves were fit using the following equations as performed in the previous studies 36,37 : . For the data sets with nonparametric distribution, Wilcoxon signed rank test is a nonparametric test was used ( Figure 1). Paired or unpaired t-tests were for used only compare the datasets obtained with and without CNS4 dose response curves. Dose dependent increase in current response was fitted into a sigmoidal curve using appropriate fitting algorithms.

| Current-voltage (I-V) relationship experiments
There are two sets of recording solutions used for the I-V assay: solutions with normal (1) and low (2)  To study the effects of CNS4 in the absence of extracellular Na + , NaCl was replaced with impermeable agent, N-methyl-D-glucamine (NMDG, 230 mM). HCl was used to dissolve the NMDG and to adjust the PH to 7.2. NMDG-containing solutions are termed Na free . The I-V relationship was studied using Xenopus oocytes expressing 1/2A and 1/2D receptors using 100 μM glutamate + 100 μM glycine agonists (with or without 100 μM CNS4) application at different holding potentials starting from −90 mV up to +40 mV in 10 mV intervals. There were four different agonist solutions used for each set of Ca 2+ recordings: (1) Agonist, (2) Agonist+CNS4, (3) Agonist in Na free solution, (4) Agonist+CNS4 in Na free solution. Each solution was applied to the oocytes in sequential order and the same order was followed for all I-V recordings. Low Ca 2+ experiments were carried out in the same way as normal Ca 2+ experiments, except that they had 0.018 mM Ca 2+ in all four agonist and recording solutions. Steady state current responses evoked by these solutions at each voltage step were measured.
Reversal potentials (E rev ) were obtained from x-intercept values when y = 0 after fitting the I-V data using a third order polynomial, nonlinear curve fitting equation Y = B0 + B1 * X + B2 * X 2 + B3 * X 3 . E rev obtained from each oocyte with different agonists were compared in a pairwise manner. These differences were then averaged. Paired or unpaired student t-test was performed to compare the data as mentioned in each figure legend.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, 38  It was noticed from the traces that a transient and completely reversible peak was observed when CNS4 alone (in the external solution) was pre-applied both in 1/2A and 1/2AB receptors ( Figure 1A,C). This unexplained peak appeared when agonist was present in one of the lines of the perfusion system. Therefore, we have hypothesized that this peak resulted from leakage of agonist solution into the tip of the CNS4-alone solution dispensing line or an artifact. However, this peak was reproducibly smaller with 1/2A receptors compared to 1/2AB receptors ( Figure 1A,C). Indeed, this peak was as strong as the agonist-induced peak in the 1/2AB receptors when the recording was repeated on the same cell multiple times. To re-confirm that this peak appears only because of agonist spillover, we designed another experiment that did not contain an agonist-alone dispensing line in the perfusion system. In this experiment, the perfusion system had only three active lines. (1) External (recording) solution, (2) 100 μM CNS4 alone, and (3) 100 μM CNS4 + 100 μM agonist solution. Other lines of the 8-channel perfusion system were physically blocked with an endcap. We expected that, since there was no agonist-alone line in this set of experiments, CNS4-alone pre-application should not generate an additional peak.
In contrast to the expectation, the peak still appeared in both  Figure 1I). However, in 1/2AB receptors, this peak was as high as 80.15 ± 2.3% of maximal current response. Nonetheless, these peaks were smaller than the ones that appeared when the agonistalone line was present ( Figure 1C). And 100 μM CNS4 did not produce any measurable current response in untransfected HEK293T cells ( Figure 1J). Therefore, currents induced by CNS4 alone are

| CNS4 does not alter competitive antagonists but alters uncompetitive NMDA receptor antagonist activity
The reduction in glycine and glutamate efficacy directed us to study the influence of CNS4 on glycine and glutamate site com-    (7) for normal & low Ca 2+ assays, respectively. E rev obtained from each oocyte with different agonists were compared in a pairwise manner. Paired student t-test was performed to compare the data presented at Ci. Unpaired student's t-test was performed to compare the difference in reversal potentials (triangles). *p < .05, **p < .01, ***p < .001, ****p < .001. WO, −18.16 ± 5.09, n = 4, versus W, −32.93 ± 7.03 mV, n = 4, p = .16.
Next, we calculated the differences in the reversal potentials (∆E rev ) between WO and W CNS4 of Na + and Na free data sets and compared them. A pairwise comparison was performed for E rev obtained from different solutions on each oocyte studied. These differences (∆E rev with Na + , 24.85 ± 3.26, n = 4 vs. Na free , −6.79 ± 1.98 mV, n = 3, p = .007) were highly significant (Figure 5Cii). Further, more negative membrane potentials (−70 mV and less) exhibited a strong inhibition of inward current, and less negative potentials (−50 mV and more) started outward rectifying. With the Na free solutions, both in the presence and absence of CNS4, maximum inward current was observed at −60 mV, as it was with the Na + solution ( Figure 5A). 378. Next, we calculated the ∆E rev between WO and W CNS4 of Na + and Na free data sets and compared them. ∆E rev with Na + , −0.22 ± 2.08, n = 4 vs Na free , −2.21 ± 2.14 mV, n = 4, p = .531

Voltage dependent changes with inward current observed in the
were not significant (Figure 6Cii).

Collectively, the results of experiments I-V indicate that CNS4
preferentially alters E rev of 1/2A currents to less negative (or positive) in the presence of Na + , at normal Ca 2+ levels. In the absence of Na + , E rev remained unchanged in both 1/2A and 1/2D receptors, indicating that CNS4 is primarily modulating Na + inward currents.
Comparison of ∆E rev revealed that Na free experiments were less impacted by CNS4 at both normal and low Ca 2+ levels in both 1/2A & 1/2D receptors (Figures 5Cii&iii and 6Cii&iii). However, in 1/2D receptors, ∆E rev were less negative at low Ca 2+ levels with Na free solutions, unlike 1/2A receptors, where equivalent ∆E rev were more negative (Figure 5Cii&iii), indicating that CNS4 maintained inward currents until the membrane potential was closer to zero (−6.22 ± 4.08 mV) in 1/2D receptors at low Ca 2+ and Na free conditions. A transient peak that occurs soon after stopping agonist application shows disinhibition of CNS4 effect (Figure 6Biii).

| Role of GluN1/2A ABD interface in determining the modulatory effect of CNS4
NMDA receptor allosteric modulators bind at various locations in the extracellular NTD or ABD interface or at pre-helices formed around the channel pore, as recently reviewed. 41 (7) for normal & low Ca 2+ assays respectively. E rev obtained from each oocyte with different agonists were compared in a pairwise manner. Paired student t-test was performed to compare the data presented at Ci. Unpaired student's t-test was performed to compare the difference in reversal potentials (triangles). *p < .05.
ABD heterodimer, preceding the pre-M1 helix positioned parallel to the lipid bilayer. 42,44 Remarkably, neither glycine nor glutamate potency was altered in E781A_1/2A receptors. 43 In contrast, another mutation at the N521 position, N521D, increased both glycine and glutamate potency compared to the wild type 1/2A receptor, as previously reported. 43 Therefore, we studied the effect of CNS4 on the N521D mutation. Interestingly, CNS4 significantly reduced gluta-

| DISCUSS ION
In the present study, we have carried out a pharmacological characterization of CNS4 which is known to potentiate 1/2C and 1/2D subtype of NMDA receptors at low glutamate (0.3 μM) concentration. 40 In the patch clamp experiments, the reproducible occurrence of a reversible peak produced by CNS4 with no agonist indicates ambient agonist sensitization (Figure 1 (Figure 4). We chose a less potent racemic mixture of AP5, DL-AP5, 45 with an expectation that even if CNS4 produced a mild effect, there would be enough room to notice this effect. Because DL-AP5 blocks only ~39% and ~18% of current in 1/2A and 1/2D receptors, there was enough current left to block (or potentiate by unblocking), if CNS4 had such a power to exercise. In contrast, CNS4 had no effect on DL-AP5 activity ( Figure 4A). This reaffirms that CNS4 is an allosteric modulator, and has no effect even on a weak orthosteric antagonist activity.
As an allosteric modulator, CNS4 was not expected to affect the activity of uncompetitive antagonist memantine that binds at   GluN1 K531A, Y535A and E781A mutants made memantine more potent, while N521A & N521D had no effect. 43 However, memantine is an uncompetitive NMDA receptor antagonist binding at the extracellular vestibule of the channel pore. 47 So, drugs binding at locations downstream from the ABD interface also can alter the 1/2A ABD interface interactions. Therefore, positive and negative modulatory effects seen with E781A and N521D are not necessarily confirming that CNS4 is binding at the ABD interface. Further mutagenesis or cryo-EM studies are needed to identify the CNS4 binding site. The visual abstract illustrates the mechanism of action of CNS4 that we hypothesize based on the observations and inference made from these results. This needs to be experimentally evaluated.
Regardless of the binding site, as long as a compound could im-  48 Thus, CNS4 can help estimate an approximate percentage of 1/2AB triheteromeric receptor currents from an animal brain tissue. In conclusion, CNS4 and its future analogs will serve as potential lead compounds to develop drugs for the treatment of neuropsychiatric conditions evolving from hypoglutamatergic conditions. Also, could be useful as chemical tools to study native NMDA receptors.
Sensitization of ambient levels of agonists in 1/2AB triheteromeric receptors, specific changes in glycine and glutamate efficacy, and modulation of permeant cation at GluN1/2 subtype are novel findings resulting from the pharmacological characterization conducted in this study. Additionally, we have identified the role of the E781 amino acid position in the activity of CNS4. This finding adds an important piece to the puzzle of understanding the molecular basis of CNS4's mechanism of action and provides insight for future drug development efforts. However, it is important to acknowledge the limitations of our study. We were unable to perform higher resolution electrophysiology assays and co-crystallization of CNS4 with one of the GluN1/2 receptors due to constraints such as lack of expertise and resources. While these experiments would have provided deeper insights into the molecular mechanisms involved, their absence does not undermine the significance of our findings.
Instead, it highlights avenues for future collaborative work to address these gaps in knowledge.
To bridge the gap between a hit compound and a lead compound, we are currently engaged in pharmacokinetics work. This ongoing research aims to determine crucial pharmacokinetic parameters, including drug absorption, bioavailability, time taken to reach maximum plasma concentration, and blood-brain barrier penetration.
The knowledge gained from these studies will be instrumental in optimizing CNS4 and its congeners to transform them into clinically valuable drug candidates. Overall, the results presented in this manuscript contribute novel insights into CNS4 pharmacology and its allosteric modulation. While some limitations exist, we believe that our findings pave the way for further investigations and potential therapeutic applications of CNS4.

AUTH O R CO NTR I B UTI O N S
Blaise M. Costa contributed to conceptualization, project administration, and manuscript writing. Blaise M. Costa and Pamela J.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors declare that all the data supporting the findings of this study are contained within the paper.